The increasing prevalence of drug-resistant bacterial infections highlights the critical medical need for new agents that are not susceptible to existing resistance mechanisms. Few new agents are in development for Gram-negative bacteria, which take up small molecules sparingly and efflux most compounds that reach the periplasm. A particularly problematic group are the multi-drug-resistant (MDR) Gram-negatives including Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Treatment of infections by these pathogens is complicated by acquired and intrinsic multi-drug resistances. The overall goal of this proposal is to address this critical medical need by discovering novel classes of antibacterials that are not subject to existing resistance mechanisms and developing them into new therapeutic or adjunctive agents for the treatment of MDR Gram-negative infections. The strategy is to focus on an unexploited essential function, lipoprotein biosynthesis which is conserved in Gram-negative bacteria and without homologs mammals. Following translocation across the inner membrane, lipoprotein precursors are acylated by lipoprotein diacylglycerol transferase (Lgt), their signal peptides cleaved off by lipoprotein signal peptidase (LspA), and further triacylated by lipoprotein N-acyl transferase (Lnt). All three enzymes are essential for viability in Gram- negative pathogens and their activity is localized to the periplasmic side of the inner membrane, indicating that inhibitors will not need to cross the inner membrane. Due to the challenges of developing high throughput biochemical screens for these targets and the need for identifying compounds that penetrate bacterial cells, target-biased whole cell screens were built in A. baumannii for both LspA and Lgt inhibitors. These consist of A. baumannii strains carrying Ptac-regulated copies of lgt and lspA in place of the chromosomal copies. Both strains cease growth and lose viability as well as cell integrity when IPTG is removed. High throughput screens were optimized based on the hypersensitivity of these strains to Lgt and LspA inhibitors in low concentrations of inducer. Both assays were validated in pilot screens against 5,000 known bioactive compounds in duplicate, yielding Z'-factors >0.7 and hit rates of ?0.1%. Moderate throughput cell-based and biochemical secondary assays of the Lgt and LspA enzymatic activities were built to validate the target specificity of hits. In Phae I, the Lgt and LspA HTS assays will be applied to >400,000 compounds, and hits will be confirmed and validated in secondary assays. Validated inhibitors will be prioritized by structure and purity, dose-dependent potency, cytotoxicity, synergy with existing antibacterials due to cell integrity effects, and bacterial spectrum including clinical isolates of P. aeruginosa, A. baumanni and carbapenem-resistant K. pneumoniae. The most potent and selective hits will be prioritized by ADME properties, mechanism of action, and SAR responsiveness to generate lead compounds. In Phase II, we will chemically optimize key scaffolds and evaluate their PK, toxicity, and efficacy in animal infection models to generate preclinical candidates.